Project Summary Gene therapy mediated by recombinant adeno-associated virus (rAAV) vectors has been clinically successful for the treatment of certain eye diseases. However, no successful clinical outcomes exist to treat hearing loss. Since the cochlea is enclosed mostly within the temporal bone, local viral gene delivery can itself cause hearing loss. In addition, the elaborate structure and exquisite function of the inner ear require coordinated action of diverse inner ear cell types, including the sensory hair cells, supporting cells, spiral ganglion neurons, and stria vascularis. No current rAAV gene delivery can specifically target spiral ganglion neurons, which are the only neuron type to carry auditory information from hair cells to central auditory system. Therefore, it would be ideal if rAAVs can target spiral ganglion neurons after a systemic injection. AAV9 has recently been found to be able to cross the blood?brain barrier after a systemic injection. Based on our previous studies and discussions with potential consumers, in this project, we proposed to make two products: (1) one new AAV9- based library with random sequences in Cap open reading frames, and (2) at least one rAAV specifically targeting spiral ganglion neurons. To achieve these goals, we have set up specific milestones in our two aims. In Aim 1, we will make a novel AAV9-based vector in which the expression of capsid proteins is coupled (but not fused) to green fluorescent protein, and then we will make random mutagenesis of the Cap gene to generation of the rAAV viral library. In Aim 2, our collaborators will inject this rAAV viral library into the mouse tail vein and isolate specific rAAV DNA from spiral ganglion neurons two weeks post injection. The process will be iterated to enhance the targeting specificity of rAAV capsid protein variants to SGNs. The novel parts of our technology are: (1) using a strategy to co-express GFP with rAAV capsid proteins, which allows direct recovery of cell-specific rAAV Cap open reading frame; (2) focusing on AAV9, which can cross the blood-brain barrier after a systemic injection; and (3) working with our collaborators, spiral ganglion neurons will be targeted without requirement of pre- purification and pre-labelling. If successful, this project will not only establish a neuronal specific targeting tool for cochlear gene therapy, but also provide an rAAV library for screening other rAAVs able to target other types of cells in the auditory system. Thus, this project will not only lead to a new rAAV vector to treat hearing loss, but also provide a new rAAV library for the research field.